Funding for work described herein was provided by the federal government, which has certain rights in the invention.
Vitamin K-dependent proteins contain 9 to 13 gamma-carboxyglutamic acid residues (Gla) in their amino terminal 45 residues. The Gla residues are produced by enzymes in the liver that utilize vitamin K to carboxylate the side chains of glutamic acid residues in protein precursors. Vitamin K-dependent proteins are involved in a number of biological processes, of which the most well-described is blood coagulation (reviewed in Furie, B. and Furie, B. C., 1988, Cell, 53:505-518). Vitamin K-dependent proteins include protein Z, protein S, prothrombin, factor X, factor IX, protein C, factor VII and Gas6. The latter protein functions in cell growth regulation. Matsubara et al., 1996, Dev. Biol., 180:499-510. The Gla residues are needed f6r proper calcium binding and membrane interaction by these proteins. The membrane contact site of factor X is thought to reside within amino acid residues 1-37. Evans and Nelsestuen, 1996, Protein Science 5: suppl. 1, 163 Abs. Although the Gla-containing regions of the plasma proteins show a high degree of sequence homology, they have at least a 1000-fold range in membrane affinity. McDonald, J. F. et al., 1997, Biochemistry, 36:5120-5137.
Factor VII functions in the initial stage of blood clotting and may be a key element in forming blood clots. The inactive precursor, or zymogen, has low enzyme activity that is greatly increased by proteolytic cleavage to form factor VIIa. This activation can be catalyzed by factor Xa as well as by VIIa-tissue factor, an integral membrane protein found in a number of cell types. Fiore, M. M., et al., 1994, J. Biol. Chem., 269:143-149. Activation by VIIa-tissue factor is referred to as autoactivation. It is implicated in both the activation (formation of factor VIIa from factor VII) and the subsequent activity of factor VIIa. The most important pathway for activation in vivo is not known. Factor VIIa can activate blood clotting factors IX and X.
Tissue factor is expressed at high levels on the surface of some tumor cells. A role for tissue factor, and for factor VIIa, in tumor development and invasion of tissues is possible. Vrana, J. A. et al., Cancer Res., 56:5063-5070. Cell expression and action of tissue factor is also a major factor in toxic response to endotoxic shock. Dackiw, A. A. et al., 1996, Arch. Surg., 131:1273-1278.
Protein C is activated by thrombin in the presence of thrombomodulin, an integral membrane protein of endothelial cells. Esmon, N. L. et al., 1982, J. Biol. Chem., 257:859-864. Activated protein C (APC) degrades factors Va and VIIIa in combination with its cofactor, protein S. Resistance to APC is the most common form of inherited thrombosis disease. Dahlback, B., 1995, Blood, 85:607-614. Vitamin k inhibitors are commonly administered as a prophylaxis for thrombosis disease.
Vitamin k-dependent proteins are used to treat certain types of hemophilia. Hemophilia A is characterized by the absence of active factor VIII, factor VIIIa, or the presence of inhibitors to factor VIII. Hemophilia B is characterized by the absence of active factor IX, factor IXa. Factor VII deficiency, although rare, responds well to factor VII administration. Bauer, K. A., 1996, Haemostasis, 26:155-158, suppl. 1. Factor VIII replacement therapy is limited due to development of high-titer inhibitory factor VIII antibodies in some patients. Alternatively, factor VIIa can be used in the treatment of hemophilia A and B. Factor IXa and factor VIIIa activate factor X. Factor VIIa eliminates the need for factors IX and VIII by activating factor X directly, and can overcome the problems of factor IX and VIII deficiencies with few immunological consequences. Hedner et al., 1993, Transfus. Medi. Rev., 7:78-83; Nicolaisen, E. M. et al., 1996, Thromb. Haemost., 76:200-204. Effective levels of factor VIIa administration are often high (45 to 90 xcexcg/kg of body weight) and administration may need to be repeated every few hours. Shulmav, S. et al., 1996, Thromb. Haemost., 75:432-436.
A soluble form of tissue factor (soluble tissue factor or sTF) that does not contain the membrane contact region, has been found to be efficacious in treatment of hemophilia when co-administered with factor VIIa. U.S. Pat. No. 5,504,064. In dogs, sTF was shown to reduce the amount of factor VIIa needed to treat hemophilia. Membrane association by sTF-VIIa is entirely dependent on the membrane contact site of factor VII. This contrasts to normal tissue-factor VIIa complex, which is bound to the membrane through both tissue factor and VII(a).
It has been discovered that modifications within the xcex3-carboxyglutamic acid (GLA) domain of vitamin K-dependent polypeptides enhance their membrane binding affinities. Vitamin K-dependent polypeptides modified in such a manner have enhanced activity and may be used as anti-coagulants, pro-coagulants or for other functions that utilize vitamin k-dependent proteins. For example, an improved factor VII molecule may provide several benefits by lowering the dosage of VIIa needed, the relative frequency of administration and/or by providing qualitative changes that allow more effective treatment of deficiency states.
The invention features vitamin k-dependent polypeptides that include a modified GLA domain that enhances membrane binding affinity of the polypeptide relative to a corresponding native vitamin k-dependent polypeptide. The modified GLA domain is from about amino acid 1 to about amino acid 45 and includes at least one amino acid substitution. For example, the amino acid substitution can be at amino acid 11, 12, 29, 33 or 34. Preferably, the substitution is at amino acid 11, 33, or 34. The modified GLA domain may include an amino acid sequence which, in the calcium saturated state, forms a tertiary structure having a cationic core with a halo of electronegative charge.
The vitamin k-dependent polypeptide may be, for example, protein C, activated protein C, factor IX, factor IXa, factor VII, factor VIIa or active site modified factor VIIa. The modified GLA domain of protein C or activated protein C may include a glutamic acid residue at amino acid 33 and an aspartic acid residue at amino acid 34. The modified GLA domain of protein C or activated protein C may also include a glutamine or glutamic acid residue at amino acid 11. Additionally, a glycine residue may be substituted at amino acid 12 in the GLA domain of protein C or activated protein C.
The modified GLA domain of factor VII, factor VIIa, active site modified factor VIIa, factor IX, and factor IXa may contain a substitution at amino acid 11, 29, 33, or combinations thereof. For example, the modified GLA domain may contain substitutions at residues 11 and 29, 11 and 33, 29 and 33, or 11, 29, and 33. The modified GLA domain can contain, for example, a substitution of a glutamine, a glutamic acid, an aspartic acid, or an asparagine residue at residue 11, and further can include a substitution at residue 29 such as substitution of a glutamic acid or a phenylalanine residue or an amino acid substitution at residue 33 such as a glutamic acid or an aspartic acid residue. The modified GLA domain can include a substitution of an aspartic acid residue at residue 33. Substitution of a glutamine residue at residue 11 is particularly useful. For example, a glutamine residue at residue 11 and a glutamic acid residue at residue 33 or a phenylalanine at residue 29 may be substituted. The GLA domain can include, for example, a substitution of a glutamic acid or a phenylalanine residue at residue 29 and further can include a substitution of a glutamic acid or an aspartic acid at residue 33. Such a polypeptide further can include a glutamic acid or an aspartic acid residue at amino acid 33.
Isolated nucleic acid molecules that include a nucleic acid sequence encoding modified vitamin K-dependent polypeptides also are described. The nucleic acid molecules encode vitamin K-dependent polypeptides that include a modified GLA domain that enhances membrane binding affinity of the polypeptide relative to a corresponding native vitamin K-dependent polypeptide. The modified GLA domain of such encoded polypeptides can include a substitution at amino acid 11, 29, or 33 as discussed above. The native vitamin K-dependent polypeptide can be, for example, factor VII, factor VIIa, active-site modified factor VIIa, factor IX, or factor IXa.
The invention also features a mammalian host cell that includes a vitamin k-dependent polypeptide. The polypeptide includes a modified GLA domain that enhances membrane binding affinity of the polypeptide relative to a corresponding native vitamin k-dependent polypeptide. The modified GLA domain includes at least one amino acid substitution at, for example, amino acid 11, 12, 29, 33 or 34. The vitamin k-dependent polypeptide may be, for example, factor VII or factor VIIa.
The invention also relates to a pharmaceutical composition that includes a pharmaceutically acceptable carrier and an amount of a vitamin k-dependent polypeptide effective to inhibit clot formation in a mammal. The vitamin k-dependent polypeptide includes a modified GLA domain that enhances membrane binding affinity of the polypeptide relative to a corresponding native vitamin k-dependent polypeptide. The modified GLA domain includes at least one amino acid substitution. The vitamin k-dependent polypeptide may be, for example, protein C, activated protein C or active site modified factor VIIa.
The invention also features a pharmaceutical composition that includes a pharmaceutically acceptable carrier and an amount of a vitamin k-dependent polypeptide effective to increase clot formation in a mammal. The vitamin k-dependent polypeptide includes a modified GLA domain that enhances membrane binding affinity of the polypeptide relative to a corresponding native vitamin k-dependent polypeptide. The modified GLA domain includes at least one amino acid substitution. For example, residue 11, 29, or 33 can be modified as discussed above. The vitamin k-dependent polypeptide may be, for example, factor VII, factor VIIa, factor IX or factor IXa. The pharmaceutical composition also may include soluble tissue factor. Such pharmaceutical compositions can be used to treat a bleeding disorder in a patient by administering the pharmaceutical composition to the patient.
A method of decreasing clot formation in a mammal is also described. The method includes administering an amount of a vitamin k-dependent polypeptide effective to decrease clot formation in the mammal. The vitamin k-dependent polypeptide includes a modified GLA domain that enhances membrane binding affinity of the polypeptide relative to a corresponding native vitamin k-dependent polypeptide. The modified GLA domain includes at least one amino acid substitution. The vitamin k-dependent polypeptide may be, for example, protein C, activated protein C or active site modified factor VIIa.
The invention also features a method of increasing clot formation in a mammal. The method includes administering an amount of a vitamin k-dependent polypeptide effective to increase clot formation in the mammal. The vitamin k-dependent polypeptide includes a modified GLA domain that enhances membrane binding affinity of the polypeptide relative to a corresponding native vitamin k-dependent polypeptide. The modified GLA domain includes at least one amino acid substitution. For example, the modified GLA domain can include an amino acid substitution at residue 11, 29, or 33, as discussed above. The vitamin k-dependent polypeptide may be, for example, factor VII, factor VIIa, factor IX or factor IXa.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.